A flow cytometric assay of murine erythrocyte osmotic fragility

Abstract

Flow cytometric test of erythrocyte osmotic fragility (EOF) is a critical diagnostic tool for evaluating membrane stability in hemolytic disorders and storage lesions of human erythrocytes. However, there is a lack of such protocols tailored for mice, one of the most widely used preclinical animal models. In this work, we developed and optimized a flow cytometric assay of murine EOF, which achieved results comparable to the conventional osmotic fragility test. We also validated the protocol by detecting membrane instability of murine erythrocytes induced by Ca² ⁺ -treatment, long-term storage, and phenylhydrazine (PHZ)-induced acute anemia, respectively. Our results showed that Ca² ⁺ -treatment impaired membrane integrity of mouse erythrocytes, resulting in reduced percentage of residual cells compared to the untreated ones (46.5 ± 9.5% vs 79.6 ± 5.3%, P  < 0.0001). We found that murine erythrocytes can be stored in 1 x PBS containing 2% FBS at 4°C for one week with intact membrane stability (81.6 ± 4.6% of residual cells vs 82.1 ± 6.0% for the fresh samples, p > 0.05). In PHZ induced anemia model, significant hemolysis was observed at day 3 post injection. In conclusion, our flow cytometric assay of murine EOF is a powerful high-throughput assay for mouse models of membrane disorders, and holds substantial scientific and translational value for investigating pathoetiological mechanisms and developing therapeutic interventions.